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• W2774278044 abstract "•PKCζ activates the MEK-ERK1/2 pathway by FGF4 stimulation•O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ activation in ESCs•FGF4-PKCζ-MEK-ERK1/2 pathway is inhibited by O-GlcNAc on PKCζ in ESCs Mouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ. Mouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ. Mouse embryonic stem cells (ESCs) are pluripotent stem cells derived from preimplantation embryos (Evans and Kaufman, 1981Evans M.J. Kaufman M.H. Establishment in culture of pluripotential cells from mouse embryos.Nature. 1981; 292: 154-156Crossref PubMed Scopus (6466) Google Scholar). They maintain the undifferentiated state via several intracellular signaling components (signal transducer and activator of transcription 3 [STAT3], extracellular signal-regulated kinase 5 [ERK5], and β-CATENIN) (Weinberger et al., 2016Weinberger L. Ayyash M. Novershtern N. Hanna J.H. Dynamic stem cell states: naïve to primed pluripotency in rodents and humans.Nat. Rev. Mol. Cell Biol. 2016; 17: 155-169Crossref PubMed Scopus (375) Google Scholar, Morikawa et al., 2016Morikawa M. Koinuma D. Mizutani A. Kawasaki N. Holmborn K. Sundqvist A. Tsutsumi S. Watabe T. Aburatani H. Heldin C.-H.H. et al.BMP sustains embryonic stem cell self-renewal through distinct functions of different Krüppel-like factors.Stem Cell Reports. 2016; 6: 64-73Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In contrast, phosphorylated ERK1/2 induces ESC differentiation (Lanner and Rossant, 2010Lanner F. Rossant J. The role of FGF/Erk signaling in pluripotent cells.Development. 2010; 137: 3351-3360Crossref PubMed Scopus (290) Google Scholar). ERK1/2 is phosphorylated (activated) by phosphorylated mitogen-activated protein kinase/extracellular signal-related kinase (MEK). Therefore, in ESCs, the MEK-ERK1/2 pathway is inhibited to maintain the undifferentiated state. However, the inhibition mechanism of the MEK-ERK1/2 pathway in ESCs is not fully understood (Li et al., 2012Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. et al.BMP4 signaling acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Chappell et al., 2013Chappell J. Sun Y. Singh A. Dalton S. MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7.Genes Dev. 2013; 27: 725-733Crossref PubMed Scopus (50) Google Scholar). O-Linked β-N-acetylglucosamine (O-GlcNAc) functions in the O-β-glycosidic attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins (Torres and Hart, 1984Torres C.R. Hart G.W. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc.J. Biol. Chem. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar). O-GlcNAc modification is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). OGT catalyzes the addition of a single O-GlcNAc residue from the donor uridine diphosphatide GlcNAc to the serine and threonine residues of core proteins (Haltiwanger et al., 1992Haltiwanger R.S. Blomberg M.A. Hart G.W. Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar). O-GlcNAc is removed from proteins by OGA (Gao et al., 2001Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain.J. Biol. Chem. 2001; 276: 9838-9845Crossref PubMed Scopus (517) Google Scholar). O-GlcNAc modification competes with phosphorylation because OGT catalyzes the addition of O-GlcNAc at or in proximity to phosphorylation sites (Zeidan and Hart, 2010Zeidan Q. Hart G.W. The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways.J. Cell. Sci. 2010; 123: 13-22Crossref PubMed Scopus (240) Google Scholar). Therefore, O-GlcNAc is believed to regulate signaling pathways by inhibiting the phosphorylation of their cytoplasmic components. Several studies have reported the functions of O-GlcNAc in ESCs. OGT is required for ESC viability; Ogt knockout mice die during embryogenesis (Watson et al., 2014Watson L.J. Long B.W. DeMartino A.M. Brittian K.R. Readnower R.D. Brainard R.E. Cummins T.D. Annamalai L. Hill B.G. Jones S.P. Cardiomyocyte Ogt is essential for postnatal viability.Am. J. Physiol. Heart Circ. Physiol. 2014; 306: H142-H153Crossref PubMed Scopus (52) Google Scholar, O’Donnell et al., 2004O’Donnell N. Zachara N. Hart G. Marth J. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.Mol. Cell. Biol. 2004; 24: 1680-1690Crossref PubMed Scopus (339) Google Scholar). Embryoid body formation assays have shown that the expression levels of OGT and O-GlcNAc are reduced during ESC differentiation (Kim et al., 2009Kim H.-S. Park S. Choi Y. Kang J. Joo H. Moon W. Cho J. Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells.FEBS Lett. 2009; 583: 2474-2478Crossref PubMed Scopus (34) Google Scholar, Jang et al., 2012Jang H. Kim T.W. Yoon S. Choi S.-Y.Y. Kang T.-W.W. Kim S.-Y.Y. Kwon Y.-W.W. Cho E.-J.J. Youn H.-D.D. O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network.Cell Stem Cell. 2012; 11: 62-74Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Furthermore, a recent study has indicated that OCT4 (POU5F1) is modified by O-GlcNAc, and that this modification regulates transcriptional activity to maintain the undifferentiated state of ESCs (Jang et al., 2012Jang H. Kim T.W. Yoon S. Choi S.-Y.Y. Kang T.-W.W. Kim S.-Y.Y. Kwon Y.-W.W. Cho E.-J.J. Youn H.-D.D. O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network.Cell Stem Cell. 2012; 11: 62-74Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). ESC differentiation into cardiac and neural cells is impaired by increased O-GlcNAc levels (Kim et al., 2009Kim H.-S. Park S. Choi Y. Kang J. Joo H. Moon W. Cho J. Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells.FEBS Lett. 2009; 583: 2474-2478Crossref PubMed Scopus (34) Google Scholar, Speakman et al., 2014Speakman C.M. Domke T.C. Wongpaiboonwattana W. Sanders K. Mudaliar M. van Aalten D.M. Barton G.J. Stavridis M.P. Elevated O-GlcNAc levels activate epigenetically repressed genes and delay mouse ESC differentiation without affecting naïve to primed cell transition.Stem Cells. 2014; 32: 2605-2615Crossref PubMed Scopus (39) Google Scholar). Our previous study demonstrated that O-GlcNAc is required for reversion from ESC-derived epiblast stem cells (ESD-EpiSCs), which correspond to the epiblast in postimplantation embryos, to ESCs (Miura and Nishihara, 2016Miura T. Nishihara S. O-GlcNAc is required for the survival of primed pluripotent stem cells and their reversion to the naïve state.Biochem. Biophys. Res. Commun. 2016; 480: 655-661Crossref PubMed Scopus (12) Google Scholar). This suggested that O-GlcNAc maintains the undifferentiated state of ESCs and that reduced O-GlcNAc is required for differentiation. However, the relation between O-GlcNAc and signaling in ESCs remains unclear. In this study, to clarify the mechanism underlying the maintenance of the undifferentiated state, we analyzed the regulation of the signaling pathways associated with differentiation via O-GlcNAc in ESCs. Here, we describe the mechanism for the maintenance of the undifferentiated state of ESCs. O-GlcNAc is reduced during embryoid body formation from ESCs (Kim et al., 2009Kim H.-S. Park S. Choi Y. Kang J. Joo H. Moon W. Cho J. Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells.FEBS Lett. 2009; 583: 2474-2478Crossref PubMed Scopus (34) Google Scholar, Jang et al., 2012Jang H. Kim T.W. Yoon S. Choi S.-Y.Y. Kang T.-W.W. Kim S.-Y.Y. Kwon Y.-W.W. Cho E.-J.J. Youn H.-D.D. O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network.Cell Stem Cell. 2012; 11: 62-74Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), suggesting that O-GlcNAc inhibits key signaling molecules that contribute to the differentiation from ESCs. Additionally, activation of the MEK-ERK1/2 pathway promotes ESC differentiation (Lanner and Rossant, 2010Lanner F. Rossant J. The role of FGF/Erk signaling in pluripotent cells.Development. 2010; 137: 3351-3360Crossref PubMed Scopus (290) Google Scholar). Phosphorylated MEK phosphorylates ERK1/2, which induces ESC differentiation. Therefore, we hypothesized that O-GlcNAc inhibits MEK and/or ERK1/2 phosphorylation in ESCs to maintain the undifferentiated state. We then performed a knockdown (KD) of Ogt mRNA using RNAi in ESCs. We designed two constructs that targeted Ogt (Ogt KD 1 and Ogt KD 2, which expressed different small interfering RNAs targeting Ogt) and one construct that targeted Egfp as a negative control. At 4 days after transfection, O-GlcNAc level and Ogt expression were lower in Ogt KD cells than in control cells (Figures 1A and 1B ). Ogt KD ESCs cannot maintain the undifferentiated state (Jang et al., 2012Jang H. Kim T.W. Yoon S. Choi S.-Y.Y. Kang T.-W.W. Kim S.-Y.Y. Kwon Y.-W.W. Cho E.-J.J. Youn H.-D.D. O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network.Cell Stem Cell. 2012; 11: 62-74Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, Shi et al., 2013Shi F.-T.T. Kim H. Lu W. He Q. Liu D. Goodell M.A. Wan M. Songyang Z. Ten-eleven translocation 1 (Tet1) is regulated by O-linked N-acetylglucosamine transferase (Ogt) for target gene repression in mouse embryonic stem cells.J. Biol. Chem. 2013; 288: 20776-20784Crossref PubMed Scopus (111) Google Scholar). The morphology of the control cells was compact and dome-shaped, similar to undifferentiated ESCs. In contrast, Ogt KD cells were flat, similar to differentiated cells (Figure 1C), indicating that the Ogt KD cells in the current study were differentiated cells. ERK1/2 phosphorylation induced GATA-binding factor 6 (GATA6) expression, which in turn inhibited NANOG expression (Figure 1D) (Chazaud et al., 2006Chazaud C. Yamanaka Y. Pawson T. Rossant J. Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway.Dev. Cell. 2006; 10: 615-624Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). GATA6- and NANOG-positive cells function as primitive endoderm (PrE)-progenitor and epiblast-progenitor cells, respectively, in mouse embryonic development at embryonic day 3.5 (E3.5) (Chazaud et al., 2006Chazaud C. Yamanaka Y. Pawson T. Rossant J. Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway.Dev. Cell. 2006; 10: 615-624Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar). Phosphorylated ERK1/2 inhibits T-box transcription factor 3 (TBX3) expression, which enhances NANOG expression (Niwa et al., 2009Niwa H. Ogawa K. Shimosato D. Adachi K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells.Nature. 2009; 460: 118-122Crossref PubMed Scopus (676) Google Scholar). ERK1/2 and MEK phosphorylation was significantly higher and NANOG expression was significantly lower in Ogt KD cells (Figures 1E–1G). OCT4 and SOX2, which are other markers of the undifferentiated state, were also significantly downregulated in Ogt KD cells (Figures S1A and S1B). These results indicated that O-GlcNAc inhibits MEK and/or ERK1/2 phosphorylation to maintain the undifferentiated state. In Ogt KD cells, Tbx3 expression was significantly decreased, and Gata6 expression was significantly increased relative to control cells (Figures 1E–1G). These results demonstrated that Ogt KD cells spontaneously differentiated into PrE cells, even in the presence of leukemia inhibitory factor (LIF). In ESCs, ERK1/2 phosphorylation is inhibited by dual-specificity phosphatase 9 (DUSP9), which is induced by bone morphogenetic protein 4 (BMP4) signaling (Figure S1C) (Li et al., 2012Li Z. Fei T. Zhang J. Zhu G. Wang L. Lu D. Chi X. Teng Y. Hou N. Yang X. et al.BMP4 signaling acts via dual-specificity phosphatase 9 to control ERK activity in mouse embryonic stem cells.Cell Stem Cell. 2012; 10: 171-182Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In Ogt KD cells, the levels of phosphorylated SMAD1/5/8, which are downstream components of BMP4 signaling and induce DUSP9, were not different compared with control cells (Figure S1D). Additionally, Dusp9 expression was unchanged in Ogt KD cells (Figure S1E). These results indicated that the increase in phosphorylated ERK1/2 in Ogt KD cells was not caused by BMP4 signaling. C-RAF and B-RAF function upstream of MEK (Galabova-Kovacs et al., 2006Galabova-Kovacs G. Kolbus A. Matzen D. Meissl K. Piazzolla D. Rubiolo C. Steinitz K. Baccarini M. ERK and beyond: insights from B-Raf and Raf-1 conditional knockouts.Cell Cycle. 2006; 5: 1514-1518Crossref PubMed Scopus (71) Google Scholar). Phosphorylated C-RAF and/or B-RAF phosphorylate MEK. In the current study, the levels of phosphorylated C-RAF and phosphorylated B-RAF were not increased in Ogt KD cells (Figure S2A). Moreover, C-RAF expression was decreased in Ogt KD cells (Figures S2A and S2B), suggesting that O-GlcNAc might contribute to the stability of C-RAF. These results indicated that the increased MEK and ERK1/2 phosphorylation in Ogt KD cells was not caused by the upregulation of C-RAF or B-RAF phosphorylation. To examine whether O-GlcNAc regulates ESC differentiation into PrE cells, we induced PrE cells from ESCs by cultivation in the absence of LIF for 6 days. The morphology of PrE cells was flat (Figure 2A). The phosphorylation levels of MEK and ERK1/2 were higher in PrE cells at 6 days than in ESCs (Figure 2B). The expression levels of Nanog and Tbx3 were decreased, while that of Gata6 was increased in PrE cells at 6 days (Figures 2B and 2C). The expression level of Ogt was significantly decreased during ESC differentiation into PrE cells (Figures 2D and 2E). O-GlcNAc was also significantly decreased in PrE cells (Figure 2F). These results suggested that O-GlcNAc inhibits the differentiation into PrE cells. Next, we examined whether O-GlcNAc inhibits ERK1/2 phosphorylation during ESC differentiation into PrE cells. We used Thiamet G and PUGNAc, which are OGA inhibitors, during the induction of ESCs into PrE cells by cultivation in the absence of LIF for 4 days. In the presence of Thiamet G (Thiamet G+) and PUGNAc (PUGNAc+), O-GlcNAc increased (Figure 2G). The upregulation of ERK1/2 phosphorylation was significantly inhibited by Thiamet G and PUGNAc (Figure 2H). GATA6 and NANOG were upregulated and downregulated, respectively, in PrE cells at 4 days in the absence of Thiamet G and PUGNAc, and the upregulation of GATA6 and downregulation of NANOG were significantly inhibited by Thiamet G and PUGNAc. These results demonstrated that O-GlcNAc inhibits the differentiation into PrE cells by inhibiting the phosphorylation of the MEK-ERK1/2 pathway in undifferentiated ESCs. To examine the function of OGT in the differentiation of ESCs to PrE cells, we overexpressed Ogt in ESCs and then induced PrE cells by cultivation in the absence of LIF for 4 days. In control cells, the MEK-ERK1/2 pathway was activated, and both OGT and markers of the undifferentiated state (OCT4, SOX2, and NANOG) were downregulated in the absence of LIF (Figure S2D). In OGT-overexpressing cells, the expression of OGT was retained despite the absence of LIF. Furthermore, activation of the MEK-ERK1/2 pathway and the downregulation of markers of the undifferentiated state by depletion of LIF were inhibited in the OGT-overexpressing cells. These results indicate that OGT inhibits activation of MEK-ERK1/2 pathway and maintains the undifferentiated state of ESCs. The FGF4-MEK-ERK1/2 pathway induces ESC differentiation into PrE cells (Lanner and Rossant, 2010Lanner F. Rossant J. The role of FGF/Erk signaling in pluripotent cells.Development. 2010; 137: 3351-3360Crossref PubMed Scopus (290) Google Scholar). Therefore, we examined the response of Ogt KD cells to FGF4 stimulation. After FGF4 stimulation, the phosphorylation levels of MEK and ERK1/2 were significantly higher in Ogt KD cells than in control cells (Figure 3A). These results demonstrated that the response of Ogt KD cells to FGF4 stimulation was markedly increased compared with control cells. The MEK inhibitor, PD0325901 (PD03), significantly inhibited the enhancement of ERK1/2 phosphorylation by FGF4 stimulation in Ogt KD cells (Figure 3B). At 24 hr after PD03 addition, markers of the undifferentiated state (OCT4, SOX2, and NANOG) and GATA6 were significantly upregulated and downregulated, respectively, in Ogt KD cells (Figures 3C, 3D, S1A, and S1B), indicating that the activation of MEK-ERK1/2 pathway reduced the expression of markers of the undifferentiated state and increased GATA6 levels in Ogt KD cells. The phosphorylation levels of C-RAF and B-RAF were not increased in Ogt KD cells compared with control cells upon FGF4 stimulation (Figure S2B), indicating that O-GlcNAc directly/indirectly inhibits MEK phosphorylation, but not that of C-RAF and B-RAF, in ESCs. In Ogt KD cells, the expression levels of Fgf4 and Fgfr2c were significantly decreased (Figure S2C), indicating that the activation of the FGF4-MEK-ERK1/2 pathway in Ogt KD cells was not caused by increased Fgf4 or Fgfr2c expression. Together, these results suggest that O-GlcNAc regulates the cytoplasmic components of FGF4 signaling. Furthermore, they indicate that O-GlcNAc inhibits the FGF4-MEK-ERK1/2 pathway in ESCs and that a reduction in OGT enhances the FGF4-MEK-ERK1/2 pathway. Next, we examined whether MEK is O-GlcNAcylated and whether its phosphorylation is inhibited in ESCs. We performed precipitation assays with biotinylated succinylated wheat germ agglutinin (sWGA), a lectin that recognizes GlcNAc, and immunoprecipitation assays with an anti-O-GlcNAc antibody (RL-2), using ESC lysates (Figures S3A and S3C). MEK did not coprecipitate with biotinylated sWGA or coimmunoprecipitate with anti-O-GlcNAc antibody (Figures S3B and S3D). These results demonstrated that MEK is not modified by O-GlcNAc in undifferentiated ESCs, suggesting that O-GlcNAc inhibits the activation of upstream components of MEK, but not of C-RAF and B-RAF, in ESCs. Phosphorylated PKCζ, which is the active form, interacts directly with MEK and phosphorylates it, independent of C-RAF and B-RAF (Monick et al., 2000Monick M.M. Carter A.B. Flaherty D.M. Peterson M.W. Hunninghake G.W. Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages.J. Immunol. 2000; 165: 4632-4639Crossref PubMed Scopus (111) Google Scholar). In human pluripotent stem cells, PKCζ is a downstream component of FGF2 signaling (Kinehara et al., 2013Kinehara M. Kawamura S. Tateyama D. Suga M. Matsumura H. Mimura S. Hirayama N. Hirata M. Uchio-Yamada K. Kohara A. et al.Protein kinase C regulates human pluripotent stem cell self-renewal.PLoS One. 2013; 8: e54122Crossref PubMed Scopus (49) Google Scholar). Moreover, in rat hepatocytes, PKCζ is modified by O-GlcNAc (Robles-Flores et al., 2008Robles-Flores M. Meléndez L. García W. Mendoza-Hernández G. Lam T.T. Castañeda-Patlán C. González-Aguilar H. Posttranslational modifications on protein kinase C isozymes. Effects of epinephrine and phorbol esters.Biochim. Biophys. Acta. 2008; 1783: 695-712Crossref PubMed Scopus (30) Google Scholar). Therefore, we hypothesized that PKCζ is a putative downstream component of FGF4 signaling and that PKCζ is modified by O-GlcNAc, which in turn inhibits PKCζ phosphorylation in undifferentiated ESCs. Therefore, we examined the effect of PKCζ phosphorylation on FGF4 stimulation. PKCζ phosphorylation was significantly enhanced by FGF4 stimulation (Figures 4A and S4A). The enhancement of MEK and ERK1/2 phosphorylation by FGF4 stimulation was significantly inhibited by two different PKC inhibitors, Gö6983 and GF 109203X hydrochloride (GFX; Figures 4A and S4A). Furthermore, PKCζ coimmunoprecipitated with anti-MEK antibody, and conversely, MEK coimmunoprecipitated with anti-PKCζ antibody (Figures 4B and 5C ). In Pkcζ KD cells (Pkcζ KD 1 and Pkcζ KD 2), at 2 days after transfection, ERK1/2 phosphorylation after FGF4 stimulation was significantly reduced compared with control cells (Figures 4C and S5A–S5C). These results demonstrated that FGF4 activates the MEK-ERK1/2 pathway via PKCζ in undifferentiated ESCs (Figure 4D). PKCα, δ, and μ also phosphorylate MEK (Wen-Sheng, 2006Wen-Sheng W. Protein kinase C alpha trigger Ras and Raf-independent MEK/ERK activation for TPA-induced growth inhibition of human hepatoma cell HepG2.Cancer Lett. 2006; 239: 27-35Crossref PubMed Scopus (65) Google Scholar, Mizuguchi et al., 2011Mizuguchi H. Terao T. Kitai M. Ikeda M. Yoshimura Y. Das A.K. Kitamura Y. Takeda N. Fukui H. Involvement of protein kinase C delta/extracellular signal-regulated kinase/poly (ADP-ribose) polymerase-1 (PARP-1) signaling pathway in histamine-induced up-regulation of histamine H1 receptor gene expression in HeLa cells.J. Biol. Chem. 2011; 286: 30542-30551Crossref PubMed Scopus (46) Google Scholar, Chen et al., 2010Chen Y.-C.C. Chen Y. Huang S.-H.H. Wang S.-M.M. Protein kinase C mu mediates adenosine-stimulated steroidogenesis in primary rat adrenal cells.FEBS Lett. 2010; 584: 4442-4448Crossref PubMed Scopus (10) Google Scholar). However, in ESCs, PKCα, δ, and μ were not activated by FGF4 stimulation (Figure S6A), indicating that they are not the downstream components of FGF4 signaling in ESCs.Figure 5The Phosphorylation Site in PKCζ Is Modified by O-GlcNAc in Undifferentiated ESCsShow full caption(A) Western blot (WB) analysis using anti-PKCζ antibody for the fraction precipitated with succinylated wheat germ agglutinin (sWGA). ESCs were precipitated with biotinylated sWGA. PKCζ was coprecipitated with biotinylated sWGA.(B) Western blot (WB) analysis using anti-PKCζ antibody for the fraction immunoprecipitated (IP) with anti-O-GlcNAc antibody (RL-2). Arrowhead indicates that PKCζ coimmunoprecipitated with anti-O-GlcNAc antibody.(C) Western blot (WB) analysis using anti-PKCζ, anti-O-GlcNAc (RL-2), anti-OGT, and anti-MEK antibodies and lectin blot (LB) analysis using sWGA-HRP for the fraction immunoprecipitated (IP) with anti-PKCζ antibody. Arrowheads indicate GlcNAc on PKCζ (LB: sWGA-HRP), O-GlcNAc on PKCζ (WB: O-GlcNAc), and MEK (WB: MEK) coimmunoprecipitated with anti-PKCζ antibody.(D) Western blot (WB) analysis using anti-PKCζ, anti-OGT, and anti-O-GlcNAc (RL-2) antibodies for the fraction immunoprecipitated (IP) with anti-PKCζ antibody using Ogt KD cells at 4 days after transfection with two constructs that expressed different shRNAs targeting Ogt (Ogt KD 1 and Ogt KD 2). Arrowheads indicate OGT (WB: OGT) and O-GlcNAc on PKCζ (WB:O-GlcNAc) coimmunoprecipitated with anti-PKCζ antibody.(E) Western blot (WB) analysis using anti-PKCζ, anti-OGT, and anti-O-GlcNAc (RL-2) antibodies for the fraction immunoprecipitated (IP) with anti-PKCζ antibody using ESCs and PrE cells at 6 days after differentiation (PrE day 6). Arrowheads indicate that OGT (WB: OGT) and O-GlcNAc on PKCζ (WB: O-GlcNAc) coimmunoprecipitated with anti-PKCζ antibody.(F) Western blot analysis using antibodies against PKCζ in cells at 2 days after transfection with the expression vector of PKCζ-FLAG tag (as wild-type) and that of T410A mutant PKCζ-FLAG tag, having a mutation of Thr-410 to Ala.(G) Immunostaining using anti-PKCζ antibody in cells expressing PKCζ-FLAG tag and those expressing T410A mutant of PKCζ-FLAG. Nuclei were stained with Hoechst (blue). Scale bars, 10 μm.(H) Western blot (WB) analysis using anti-PKCζ and anti-O-GlcNAc (RL-2) antibodies for the fraction immunoprecipitated (IP) with anti-FLAG antibody. Arrowheads indicate PKCζ (WB: PKCζ) and O-GlcNAc on PKCζ (WB: O-GlcNAc) immunoprecipitated with anti-FLAG antibody. The histograms show the mean densitometric readings ± SD of (O-GlcNAc on PKCζ immunoprecipitated with anti-FLAG antibody)/(PKCζ immunoprecipitated with anti-FLAG antibody) after normalization against the levels in PKCζ-FLAG-expressing cells (set to 1).(I) Western blot (WB) analysis using anti-PKCζ, anti-O-GlcNAc (RL-2), anti-phospho-Thr-410 of PKCζ (p-PKCζ pT410), and anti-MEK antibodies for the fraction immunoprecipitated (IP) with anti-FLAG antibody using cells expressing PKCζ-FLAG tag in the presence or absence of 200 μM PUGNAc for 24 hr. Arrowheads indicate O-GlcNAc on PKCζ-FLAG (WB: O-GlcNAc) and the phosphorylation of Thr-410 of PKCζ-FLAG (WB: p-PKCζ pT410).(J) Western blot analysis using antibodies against phospho-MEK (p-MEK), MEK, phospho-ERK1/2 (p-ERK1/2), and ERK1/2 in cells expressing PKCζ-FLAG tag, cells expressing PKCζ-FLAG tag in the presence of 200 μM PUGNAc for 24 hr (PKCζ + PUGNAc), and cells expressing the T410A mutant of PKCζ-FLAG tag after FGF4 stimulation (50 ng/mL) for 3 min. The histograms show the mean densitometric readings ± SD of p-MEK/MEK and p-ERK1/2/ERK1/2 after normalization against the levels in control cells stimulated with FGF4 (set to 1). n.s., not significant.Representative images of the western blots and immunostaining are shown. Values were obtained from three independent experiments, and significant values in comparison with cells expressing PKCζ-FLAG tag (H) or control cells stimulated with FGF4 (J) are indicated as ∗p < 0.05 and ∗∗p < 0.01. Hash indicates that only anti-O-GlcNAc (B), anti-PKCζ (C), and anti-FLAG (H) antibodies were loaded to detect the heavy and light chains of the antibodies used for immunoprecipitation. Asterisk indicates the heavy chain or light chain of the antibodies used for immunoprecipitation. See also Figure S6.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Western blot (WB) analysis using anti-PKCζ antibody for the fraction precipitated with succinylated wheat germ agglutinin (sWGA). ESCs were precipitated with biotinylated sWGA. PKCζ was coprecipitated with biotinylated sWGA. (B) Western blot (WB) analysis using anti-PKCζ antibody for the fraction immunoprecipitated (IP) with anti-O-GlcNAc antibody (RL-2). Arrowhead indicates that PKCζ coimmunoprecipitated with anti-O-GlcNAc antibody. (C) Western blot (WB) analysis using anti-PKCζ, anti-O-GlcNAc (RL-2), anti-OGT, and anti-MEK antibodies and lectin blot (LB) analysis using sWGA-HRP for the fraction immunoprecipitated (IP) with anti-PKCζ antibody. Arrowheads indicate GlcNAc on PKCζ (LB: sWGA-HRP), O-GlcNAc on PKCζ (WB: O-GlcNAc), and MEK (WB: MEK) coimmunoprecipitated with anti-PKCζ antibody. (D) Western blot (WB) analysis using anti-PKCζ, anti-OGT, and anti-O-GlcNAc (RL-2) antibodies for the fraction immunoprecipitated (IP) with anti-PKCζ antibody using Ogt KD cells at 4 days after transfection with two constructs that expressed different shRNAs ta" @default.
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• W2774278044 title "O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells" @default.
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• W2774278044 doi "https://doi.org/10.1016/j.stemcr.2017.11.007" @default.
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